Porcine respiratory disease complex induces pulmonary fibrosis related to the aberrant sphingolipid metabolism

Abstract

Porcine respiratory disease complex (PRDC) is a common syndrome in the modern swine industry worldwide, and its pathogenesis remains unclear to date. Our study aimed to investigate PRDC-induced pulmonary fibrosis and sphingolipid metabolism, and their relationship. Mouse and cell line (A549 and 3D4/21) models exposed to bleomycin and/or transforming growth factor-β1 (TGF-β1) were developed. Histopathological and immunohistochemical staining, colorimetry, lipidomics analysis and pharmacologic intervention assays were used to analyse lung fibrosis and sphingolipid profiles. PRDC was validated by the presence of alveolar epithelial cell (AEC) injury and hyperplasia, inflammatory infiltrates, asymmetric macrophage polarization and mast cell phenotypic changes, TGF-β1 and fibroblast growth factor 2 (FGF-2) overproduction, extensive collagen deposition, foci of fibroblast/myofibroblast with stress fibres (α-SMA, γ-SMA and γ2 actin), cell interaction with increasing frequency, proliferation, apoptosis and autophagy dysregulation, and mucin 6 release-all of which are characteristics of pulmonary fibrosis. Based on the sphingolipidomics and pharmacologic interventions data-the dysregulated sphingolipids, including sphingomyelin (SM), ceramide (Cer), sphingosine-1-phosphate (S1P) and cerebroside (Cb), possibly due to serine palmitoyltransferase (SPT; SPTLC1), ceramide synthase (CerS; CerS2, CerS4), sphingomyelin synthase (SMS; SMS1), neutral sphingomyelinase (NSMase), acid sphingomyelinase (ASMase; SMPDL3B) and sphingosine kinase (SphK; SphK1, SphK2), were found to be closely related to pulmonary fibrosis. Furthermore, d18:1 24:1 SM and 18:1 S1P may be conserved biomarkers and tiamulin fumarate (TF) changes have anti-fibrotic activity. Overall, PRDC induces pulmonary fibrosis, related to the aberrant sphingolipid metabolism, where conserved sphingolipid biomarkers and anti-fibrotic candidates have been found.

Keywords: metabolism; porcine respiratory disease complex; pulmonary fibrosis; sphingolipid.

FIGURE 1

Porcine respiratory disease complex (PRDC) induces pulmonary fibrosis. (A) PRDC induces pulmonary fibrosis with cell survival and death paradox. The right lung lower lobes from the healthy control (HC) and PRDC pigs were stained with haematoxylin and eosin (HE), Masson's trichrome (MT) and immunohistochemical staining (IHC) of markers of proliferation (Ki67), apoptosis (CC3) and autophagy (LC3B). The staining was analysed by light microscopy, and 12 high power fields (HPFs) were examined at a magnification of ×400, followed by quantitative analysis of histology score, Ashcroft score and Cells/HPF. Lung structure distortion and alveolar architecture obliteration (arrow a1), hyperplastic epithelial cells (arrow a2), inflammatory infiltrates (arrow a3), foamy macrophage accumulation within alveolar spaces and foamy change within alveolar septa (arrow a4), myofibroblast core (arrow a5) and the active fibrotic front (arrow a6), honeycomb zone (arrow a7), fibroblastic foci (FF) (arrow a8), collagen fibres (blue, arrow a9) and Masson body (arrow a10) were present in PRDC lung parenchyma. The Ki67‐positive cells were present in FF (arrow a1), alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). The CC3‐positive cells were shown in alveolar epithelium (arrow a2). The LC3B‐positive cells were present in alveolar epithelium (arrow a2). (B) Stress fibres formation and fibrogenic mediators secretion contribute to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of stress fibres (α‐SMA, γ‐SMA and γ2 Actin) and fibrogenic mediator (TGF‐β1 and FGF‐2). The staining was analysed as described above. The positive cells were predominantly distributed within FF (arrow a1), alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). (C) Macrophage/mast cell polarization and myofibroblast–mast cells interaction contribute to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of M2 macrophage (CD68 and CD163) and mast cells (Tryptase and Chymase). The staining was analysed as described above. The positive cells (macrophages and mast cells) were predominantly distributed within airspaces and alveolar septa (black arrow). Dual‐stained Chymase (brown) and α‐SMA (pink)‐positive cells were mainly distributed within FF and pulmonary vessel walls (black arrow). (D) Mucin 6 is related to pulmonary fibrosis induced by PRDC. The right lung lower lobes from HC and PRDC pigs were stained with IHC of Mucin 6. The staining was analysed as described above. The Mucin 6‐positive cells were present in hyperplastic alveolar epithelium (arrow a2) and inflammatory infiltrates (arrow a3). (E) Hydroxyproline is positively related to pulmonary fibrosis induced by PRDC. Hydroxyproline in the lungs from HC and PRDC pigs was measured by the colorimetric assay. (F) Average daily weight gain (ADWG) is negatively related to pulmonary fibrosis induced by PRDC. ADWG of the HC and PRDC pigs was from 10 weeks of age. Representative photomicrographs from 10 pigs were shown. Nuclei were stained with haematoxylin (blue). Scale bar = 50 μm. All data were presented as mean ± SD. *p < .05, **p < .01.

FIGURE 2

Bleomycin sulphate (BMS) induces pulmonary fibrosis. On Day 21, the lungs from phosphate‐buffered saline (PBS) and BMS‐treated mice were paraformaldehyde‐fixed and stained with haematoxylin and eosin (HE), Masson's trichrome (MT) (A), immunohistochemical staining (IHC) (α‐SMA, TGF‐β1) (B). The staining was analysed as described above. The characteristics of pulmonary fibrosis were indicated by the arrow a1, a2, a3, a4, a8, a9 and a10, respectively. α‐SMA‐ and TGF‐β1‐positive staining within fibroblastic foci (FF), alveolar epithelium and inflammatory infiltrates were indicated by the arrow a11, a12 and a13, respectively. Nuclei were stained with haematoxylin (blue). The number of α‐SMA⁺ and TGF‐β1⁺ cells per high power fields (HPF) was counted. Hydroxyproline (C) in the lungs, TGF‐β1 (D) in the BALF and lungs were measured by the colorimetric assay, respectively. The representative micrographs from three independent experiments were presented. Scale bar = 50 μm. All data were presented as mean ± SD. *p < .05, **p < .01.

FIGURE 3

Porcine respiratory disease complex (PRDC) affects porcine lung sphingolipid metabolism. Sphingolipids (pmol/mg) in the upper lobes of right lungs from the healthy control (HC) and PRDC pigs were analysed with LC‐MS/MS as described in materials and methods. (A) SM, (B) Cer, (C) S1P, (D) Cb. The representative micrographs from 10 pigs were shown. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

FIGURE 4

Pulmonary fibrosis induced by porcine respiratory disease complex (PRDC) is positively related to the level of sphingolipid biomarker. The lungs from healthy control (HC), PRDC and PRDC+TF (TF)‐treated pigs were paraformaldehyde‐fixed or frozen‐fixed, stained with haematoxylin and eosin (HE), Masson's trichrome (MT) (A) and IHC (α‐SMA, TGF‐β1, A, B), and analysed by light microscopy and/or by fluorescence microscopy. Twelve high power fields (HPFs) were examined at a magnification of ×100 or ×400. The characteristics of pulmonary fibrosis were described as shown above. Positive cell was indicated by the arrow. Nuclei were stained with haematoxylin or DAPI (blue). Hydroxyproline (C) and TGF‐β1 (D) in the lungs were measured by the colorimetric assay, respectively. The sphingolipid biomarkers (d18:1 24:1 SM, 18:1 S1P) (E) in the lungs were analysed with LC‐MS/MS as described in Materials and Methods. The representative micrographs from three independent experiments were presented. Scale bar = 50 or 20 μm. The histology score, Ashcroft score, Cells/HPF, hydroxyproline (μg/mg), TGF‐β1 (pg/mg) and sphingolipid biomarker (pmol/mg) were analysed using GraphPad Prism v6.02. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

FIGURE 5

Pulmonary fibrosis induced by bleomycin sulphate (BMS) is positively related to the level of sphingolipid biomarker. On Day 21, the tissues from phosphate‐buffered saline (PBS), BMS and BMS + TF (TF)‐treated mice were paraformaldehyde‐fixed or frozen‐fixed, stained with haematoxylin and eosin (HE) (A, F), Masson's trichrome (MT) (A) and IHC (α‐SMA, B), and analysed by light microscopy and/or by fluorescence microscopy. Twelve HPFs were examined at a magnification of ×400 or ×1000. The characteristics of pulmonary fibrosis were described as shown above. Positive cell was indicated by the arrow. Nuclei were stained with DAPI (blue). As to spleen, haemorrhage, oedema, megakaryocyte, necrosis, inflammatory foci and fibroplasia were indicated by the arrow b1, b2, b3, b4, b5 and b6, respectively. Hydroxyproline (C) and TGF‐β1 (D) in the lungs were measured by the colorimetric assay, respectively. The sphingolipid biomarkers (d18:1 24:1 SM, 18:1 S1P) (E) in the lungs were analysed with LC‐MS/MS as described in materials and methods. The representative micrographs from 3 independent experiments were presented. Scale bar = 50 or 20 μm. The MFI was quantified with NIH ImageJ v1.46, and the histology score, Ashcroft score, MFI, hydroxyproline (μg/mg), TGF‐β1 (pg/mg) and sphingolipid biomarker (pmol/mg) were analysed with GraphPad Prism v6.02. All data were presented as mean ± SD. *p < .05, **p < .01, versus corresponding control.

FIGURE 6

Schematic model of the proposed sequence of events and sphingolipid metabolism leading to pulmonary fibrosis induced by porcine respiratory disease complex (PRDC). The persistent or repetitive fibrogenic stimuli are capable of inducing alveolar epithelium (AE, type I and type II) lesions, such as injury, hyperplasia, apoptosis, barrier function damage, epithelial–mesenchymal transition (EMT) and pro‐fibrotic factors (TGF‐β1, Mucin 6) secretion. These events recruit alveolar macrophage (AM), mast cell (MC) and fibroblast (FB) to the fibrogenesis centre, where proliferation, activation, M2 polarization, myofibroblast (myoFB) transdifferentiation, cell to cell crosstalk and pro‐fibrotic factors production happen to them, and further ECM expansion/ deposition, alveolar/interstitium architecture destruction, fibroblastic foci (FF) formation and final fibrosis are developed. The activation and migration of these structural cells (epithelial cells and fibroblasts) and immune cells (macrophages and mast cells)‐associated pathways, the release of pro‐fibrotic factors (cytokine, growth factor, glycoprotein, lipid and enzyme), as well as the interplays among them, play important roles in the initiation, progression and chronic development of PRDC‐induced pulmonary fibrosis. Meanwhile, fibrogenic stimuli and pro‐fibrotic injury induces metabolic stress in fibrogenesis centre, leading to abnormal sphingolipid metabolism. SPT catalyses the initial rate‐limiting step of de novo pathways of sphingolipid biosynthesis, followed by the generation of Cer. The latter is central to sphingolipid metabolism, being a substrate of different enzymes for sphingolipid biosynthesis and catabolism. Cer can be catalysed by ceramide glucosyltransferase (UGCG) and ceramide galactosyltransferase (CGT) to form Cb, and the latter can be metabolized by β‐glucocerebrosidase (GCase) to form Cer. Cer can be further metabolized into SM by SMS1. SM is then hydrolysed to form Cer by NSMase and ASMase. Cer, coming from SM, can be further degraded to Sph by several organelle‐specific ceramidases, and SphK1 or SphK2 phosphorylates Sph to S1P. In addition, Sph can be re‐acylated by CerS2 and CerS4 to Cer (salvage pathway). Dysregulation of specific metabolic enzymes and sphingolipid is closely related to pulmonary fibrosis and the events described above. See text for details.